Abstract

Background

In order to generate biofuels, insoluble cellulosic substrates are pretreated and
subsequently hydrolyzed with cellulases. One way to pretreat cellulose in a safe
and environmentally friendly manner is to apply, under mild conditions,
non-hydrolyzing proteins such as swollenin - naturally produced in low yields by
the fungus Trichoderma reesei. To yield sufficient swollenin for
industrial applications, the first aim of this study is to present a new way of
producing recombinant swollenin. The main objective is to show how swollenin
quantitatively affects relevant physical properties of cellulosic substrates and
how it affects subsequent hydrolysis.

Results

After expression in the yeast Kluyveromyces lactis, the resulting
swollenin was purified. The adsorption parameters of the recombinant swollenin
onto cellulose were quantified for the first time and were comparable to those of
individual cellulases from T. reesei. Four different insoluble cellulosic
substrates were then pretreated with swollenin. At first, it could be
qualitatively shown by macroscopic evaluation and microscopy that swollenin caused
deagglomeration of bigger cellulose agglomerates as well as dispersion of
cellulose microfibrils (amorphogenesis). Afterwards, the effects of swollenin on
cellulose particle size, maximum cellulase adsorption and cellulose crystallinity
were quantified. The pretreatment with swollenin resulted in a significant
decrease in particle size of the cellulosic substrates as well as in their
crystallinity, thereby substantially increasing maximum cellulase adsorption onto
these substrates. Subsequently, the pretreated cellulosic substrates were
hydrolyzed with cellulases. Here, pretreatment of cellulosic substrates with
swollenin, even in non-saturating concentrations, significantly accelerated the
hydrolysis. By correlating particle size and crystallinity of the cellulosic
substrates with initial hydrolysis rates, it could be shown that the
swollenin-induced reduction in particle size and crystallinity resulted in high
cellulose hydrolysis rates.

Conclusions

Recombinant swollenin can be easily produced with the robust yeast K.
lactis. Moreover, swollenin induces deagglomeration of cellulose
agglomerates as well as amorphogenesis (decrystallization). For the first time,
this study quantifies and elucidates in detail how swollenin affects different
cellulosic substrates and their hydrolysis.